High fat intake is a major environmental factor leading to decreased insulin sensitivity contributing to the rising incidence of interrelated insulin resistant diseases such as Syndrome X, PCOS, Type 2 diabetes mellitus, and obesity. We have demonstrated that estrogenized women and female rodents are protected from fat induced insulin resistance, whereas, males, and estrogen deficient females are fully susceptible to these adverse effects of fat. In this application, we plan a broad based, in vivo and in vitro approach to elucidate the mechanisms of fat induced insulin resistance and the protective effects of estrogens, using various novel animal model systems, 3T3-L1 adipocytes in vitro, and the non-classical insulin target tissue ovarian granulose cells (GCs) An underlying hypothesis in this application is that excess fat metabolism due to elevated FFA levels, or high fat diets, leads to activation of the "inflammatory pathway" and that specific serine/ threonine kinases in this pathway such as PKC theta, IKK beta, or JNK, or genes induced as a result of NfkappaB activation, feedback on the insulin signaling system to cause insulin resistance. Our preliminary data show that treatment of 3T3-L1 adipocytes in vitro with FFAs leads to a marked state of cellular insulin resistance, and we will exploit this novel system to conduct new studies aimed at elucidating the molecular mechanisms of FFA induced insulin resistance and estrogen's protection against these effects. Finally, since we hypothesize that GCs from insulin resistant animals and women (particularly PCOS) can be insulin/ IGF-I resistant with functional consequences, we propose an extensive series of studies in GCs prepared from normal rats and insulin resistant rodents, to determine whether FFA treatment causes insulin resistance in these cells, as it dos in insulin target cells, and to identify the underlying mechanisms. We will also study the basic signaling systems for insulin, IGF-I and FSH in these cells. Taken together the results of these studies should greatly enhance our understanding of the mechanisms of fat induced insulin resistance, in classic and non-classic insulin target tissues, and also elucidate the mechanisms underlying the protective effects of estrogens. These studies should also highlight the role of inflammatory pathway activation in these pathophysiologic events and this may have potential therapeutic implications for new treatment approaches.